During fiscal year 2010 we accomplished the following: 1) We initiated analyses on BLSA subjects who had returned for their 2 year follow-up visit. CD4+ T cells from 6 subjects were purified and activated with anti-CD3. Nuclear and cytoplasmic extracts were prepared and analyzed for NF-&#954;B induction, total RNA was isolated for gene expression profiling. 2) We completed analyzing 30 additional first-time BLSA subjects. 3) We carried out 2D protein electrophoresis with 4 pairs of cytosolic extracts prepared from CD4+ T cells from young and old females and 4 pairs of extracts from young and old males. We found several reproducible changes in the 2D pattern between young and old. To identify proteins that were differentially expressed we prepared cytosolic extracts from large cell numbers and carried out preparative 2D electrophoresis. Differentially expressed proteins were excised, eluted from gels and are being analyzed by mass spectroscopy. 4) We compared gene expression in 24 samples of CD4+ T cells that were kept at 4oC to the same cells cultured at 37oC for 4 h. We found cell-intrinsic activation of the NF-&#954;B pathway at 37oC and down-regulation of antigen receptor-induced gene expression. We conjectured that cells maintained at 4oC reflected the gene expression signature of 'tonic'T cell receptor signals that are essential for T cell homeostasis in vivo. Ex vivo culture disrupts TCR/MHC interactions that occur in-vivo and, instead, up-regulates NF-&#954;B target genes. To rule out activation effects of fetal calf serum (FCS) that is present in tissue-culture medium during 37oC incubation, these studies are being repeated with synthetic medium that does not contain FCS.